A novel serum free primary astrocyte culture method that mimic quiescent astrocyte phenotype

Abstract

Background: Primary astrocyte cultures have been used for decades to study astrocyte functions in health and disease. The current primary astrocyte cultures are mostly maintained in serum-containing medium which produces astrocytes with a reactive phenotype as compared to in vivo quiescent astrocytes. The aim of this study was to establish a serum-free astrocyte culture medium that maintains primary astrocytes in a quiescent state.

New method: Serum free astrocyte base medium (ABM) supplemented with basic fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF) (ABM-FGF2-EGF) or serum supplemented DMEM (MD-10%FBS) was used to culture primary astrocytes isolated from cerebral cortex of postnatal day 1 C57BL/6 mice.

Results: Compared to astrocytes cultured in MD-10%FBS medium, astrocytes in ABM-FGF2-EGF had higher process bearing morphologies similar to in vivo astrocytes. Western blot, immunostaining, quantitative polymerase chain reaction and metabolic assays revealed that astrocytes maintained in ABM-FGF2-EGF had enhanced glycolytic metabolism, higher glycogen content, lower GFAP expression, increased glutamine synthase, and glutamate transporter-1 mRNA levels as compared to astrocytes cultured in MD-10% FBS medium.

Comparison to existing methods: These observations suggest that astrocytes cultured in ABM-FGF2-EGF media compared to the usual FBS media promote quiescent and biosynthetic phenotype similar to in vivo astrocytes.

Conclusion: This media provides a novel method for studying astrocytes functions in vitro under physiological and pathological conditions.

Keywords: Astrocyte; Astrogliosis; Basic fibroblast growth factor; Epidermal growth factors; Fetal bovine serum; Primary culture.